Methods for treating pulmonary non-tuberculous mycobacterial infections

ABSTRACT

Provided herein are methods for treating a pulmonary infection in a patient in need thereof, for example, a nontuberculous mycobacterial pulmonary infection for at least one treatment cycle. The method comprises administering to the lungs of the patient a pharmaceutical composition comprising a liposomal complexed aminoglycoside comprising a lipid component comprising electrically neutral lipids and an aminoglycoside. Administration comprises aerosolizing the pharmaceutical composition to provide an aerosolized pharmaceutical composition comprising a mixture of free aminoglycoside and liposomal complexed aminoglycoside, and administering the aerosolized pharmaceutical composition via a nebulizer to the lungs of the patient. The methods provided herein result in a change from baseline on the semi-quantitative scale for mycobacterial culture for a treated patient, and/or NTM culture conversion to negative during or after the administration period.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/250,476, filed Jan. 17, 2019, which is a continuation of U.S.application Ser. No. 15/311,488, filed Feb. 7, 2017, now U.S. Pat. No.10,238,675, which is a 371 national stage entry of InternationalApplication No. PCT/US2015/031079, filed May 15, 2015, which claimspriority to U.S. Provisional Application Ser. No. 61/993,439, filed May15, 2014; 62/042,126, filed Aug. 26, 2014; 62/048,068, filed Sep. 9,2014; and 62/056,296, filed Sep. 26, 2014, the disclosure of each ofwhich is incorporated by reference herein in their entireties for allpurposes.

BACKGROUND OF THE INVENTION

Certain technologies suitable for administration by inhalation employliposomes and lipid complexes supply a prolonged therapeutic effect ofdrug in the lung. These technologies also provide the drug withsustained activities, and the ability to target and enhance the uptakeof the drug into sites of disease.

Inhalation delivery of liposomes is complicated by their sensitivity toshear-induced stress during nebulization, which can lead to change inphysical characteristics (e.g., entrapment, size). However, as long asthe changes in characteristics are reproducible and meet acceptabilitycriteria, they need not be prohibitive to pharmaceutical development.

Pulmonary infection with non-tuberculous mycobacterium (NTM) in thesusceptible host can lead to potentially severe morbidity and evenmortality among those affected. As infection rates are rising, pulmonarynontuberculous mycobacterial disease (PNTM) represents an emergingpublic health concern in the United States. NTM are ubiquitous in theenvironment. Over 80% of pulmonary NTM (PNTM) infections in the US aredue to Mycobacterium avium complex (MAC). In addition, M. kansasii, M.abscessus, and M. fortuitum are regularly isolated.

The prevalence of pulmonary NTM infections in the United States has morethan doubled in the last 15 years. The ATS/IDSA PNTM reported 2-yearperiod prevalence of pulmonary NTM infections is 8.6/100,000 persons.The prevalence of pulmonary NTM infections increases with age with20.4/100,000 in those at least 50 years of age and is especiallyprevalent in females (median age: 66 years; female: 59%).

In the susceptible individual, pulmonary NTM infections can be seriousor life threatening. Available therapies may be poorly tolerated, andmay have significant adverse events. The present invention addressesthis and other needs by providing methods for treating pulmonary NTMinfections in patients in need thereof.

SUMMARY OF THE INVENTION

The present invention, in one aspect, provides methods for treating orproviding prophylaxis against a nontuberculous mycobacterial (NTM)infection (pulmonary infection caused or due to one or morenontuberculous mycobacteria), via inhalation administration of aneffective amount of a composition comprising a liposomal complexedaminoglycoside, or a pharmaceutically acceptable salt thereof, to apatient in need thereof. The patient in need of treatment, in oneembodiment, is a cystic fibrosis patient, a bronchiectasis patient,suffers from asthma or suffers from chronic obstructive pulmonarydisorder (COPD).

In one embodiment, the NTM infection is a pulmonary NTM infectionselected from an M. avium, M. avium subsp. hominissuis (MAH), M.abscessus, M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium,M. avium complex (MAC) (M. avium and M. intracellulare), M. conspicuum,M. kansasii, M. peregrinum, M. immunogenum, M. xenopi, M. marinum, M.malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum, M.scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terraecomplex, M. haemophilum, M. genavense, M. gordonae, M. ulcerans, M.fortuitum, M. fortuitum complex (M. fortuitum and M. chelonae) infectionor a combination thereof. In a further embodiment, the NTM infection isan M. avium complex (MAC) (M. avium and M. intracellulare) infection. Inone embodiment, the NTM infection is a pulmonary recalcitrant NTMinfection.

In one embodiment, the composition comprising the liposomal complexedaminoglycoside is a dispersion (e.g., a liposomal solution orsuspension). The liposomal portion of the composition comprises a lipidcomponent that includes electrically neutral lipids. In a furtherembodiment, the electrically neutral lipids comprise aphosphatidylcholine and a sterol (e.g., dipalmitoylphosphatidylcholineand cholesterol). In a further embodiment, the aminoglycoside isamikacin or a pharmaceutically acceptable salt thereof. In even afurther embodiment, the aminoglycoside is amikacin sulfate.

In one embodiment, the method for treating or providing prophylaxisagainst an NTM infection comprises administering an aerosolizedpharmaceutical composition to the lungs of the patient in need thereof;wherein the aerosolized pharmaceutical composition comprises a mixtureof free aminoglycoside and liposomal complexed aminoglycoside, and thelipid component of the liposome consists of electrically neutral lipids.In a further embodiment, the electrically neutral lipids comprise aphosphatidylcholine and a sterol (e.g., dipalmitoylphosphatidylcholineand cholesterol). In a further embodiment, the aminoglycoside isamikacin or a pharmaceutically acceptable salt thereof. In even afurther embodiment, the aminoglycoside is amikacin sulfate.

The methods provided herein result in a change from baseline on thesemi-quantitative scale for mycobacterial culture for a treated patient,and/or NTM culture conversion to negative during or after theadministration period. For example, in one embodiment, the methodprovided herein results in the patient having an NTM culture conversionto negative after an administration period.

In one embodiment, the aminoglycoside or pharmaceutically acceptablesalt thereof is amikacin, apramycin, arbekacin, astromicin, capreomycin,dibekacin, framycetin, gentamicin, hygromycin B, isepamicin, kanamycin,neomycin, netilmicin, paromomycin, rhodestreptomycin, ribostamycin,sisomicin, spectinomycin, streptomycin, tobramycin, verdamicin, apharmaceutically acceptable salt thereof, or a combination thereof. Ineven a further embodiment, the aminoglycoside is amikacin. In anotherembodiment, the aminoglycoside is selected from an aminoglycoside setforth in Table 1, below, a pharmaceutically acceptable salt thereof, ora combination thereof.

TABLE 1 Aminoglycosides for use with the present invention AC4437dibekacin K-4619 sisomicin amikacin dactimicin isepamicinrhodestreptomycin apramycin etimicin KA-5685 sorbistin arbekacinframycetin kanamycin spectinomycin astromicin gentamicin neomycinsporaricin bekanamycin H107 netilmicin streptomycin boholmycinhygromycin paromomycin tobramycin brulamycin hygromycin B plazomicinverdamicin capreomycin inosamycin ribostamycin vertilmicin

The pharmaceutical compositions provided herein in one embodiment aredispersions of liposomes (i.e., liposomal dispersions or aqueousliposomal dispersions which can be either liposomal solutions orliposomal suspensions). In one embodiment, the lipid component of theliposomes consists essentially of one or more electrically neutrallipids. In a further embodiment, the electrically neutral lipidcomprises a phospholipid and a sterol. In a further embodiment, thephospholipid is dipalmitoylphosphatidylcholine (DPPC) and the sterol ischolesterol.

In one embodiment, the lipid to aminoglycoside weight ratio in theaminoglycoside pharmaceutical composition (aminoglycoside liposomalsolution or suspension) is about 2:1, about 2:1 or less, about 1:1,about 1:1 or less, about 0.75:1 or less, or about 0.7:1. In anotherembodiment, the lipid to aminoglycoside weight ratio in the compositionis from about 0.10:1 to about 1.25:1, from about 0.10:1 to about 1.0:1,from about 0.25:1 to about 1.25:1, from about 0.5:1 to about 1:1.

In one embodiment, the methods provided herein comprise administrationof the liposomal aminoglycoside composition via nebulization oraerosolization. The method in this embodiment therefore entailsgeneration of an aerosolized aminoglycoside composition. In oneembodiment, upon nebulization, the aerosolized composition has anaerosol droplet size of about 1 μm to about 3.8 μm, about 1.0 μm to 4.8μm, about 3.8 μm to about 4.8 μm, or about 4.0 μm to about 4.5 μm. In afurther embodiment, the aminoglycoside is amikacin. In even a furtherembodiment, the amikacin is amikacin sulfate.

In one embodiment, about 70% to about 100% of the aminoglycoside presentin the composition is liposomal complexed, e.g., encapsulated in aplurality of liposomes, prior to administration to the patient in needof treatment. In a further embodiment, the aminoglycoside is selectedfrom an aminoglycoside provided in Table 1. In further embodiment, theaminoglycoside is an amikacin (e.g., as amikacin sulfate). In even afurther embodiment, about 80% to about 100% of the amikacin is liposomalcomplexed, or about 80% to about 100% of the amikacin is encapsulated ina plurality of liposomes, prior to administration to the patient in needof treatment. In another embodiment, prior to administration to thepatient in need of treatment (i.e., prior to nebulization), about 80% toabout 100%, about 80% to about 99%, about 90% to about 100%, 90% toabout 99%, or about 95% to about 99% of the aminoglycoside present inthe composition is liposomal complexed.

In one embodiment, the percent liposomal complexed (also referred toherein as “liposomal associated”) aminoglycoside post-nebulization isfrom about 50% to about 80%, from about 50% to about 75%, from about 50%to about 70%, from about 55% to about 75%, or from about 60% to about70%. In a further embodiment, the aminoglycoside is selected from anaminoglycoside provided in Table 1. In a further embodiment, theaminoglycoside is amikacin. In even a further embodiment, the amikacinis amikacin sulfate. In one embodiment, the aerosolized composition(i.e., post nebulization) comprises from about 65% to about 75%liposomal complexed aminoglycoside and from about 25% to about 35% freeaminoglycoside. In a further embodiment, the aminoglycoside is amikacin.In even a further embodiment, the amikacin is amikacin sulfate.

In one embodiment, the pulmonary infection treated by the methodsprovided herein is a Mycobacterium abscessus pulmonary infection or aMycobacterium avium complex pulmonary infection. In one or more of thepreceding embodiments, the patient is a cystic fibrosis patient, abronchiectasis patient, an asthma patient or a COPD patient.

In one embodiment, a patient with cystic fibrosis is treated for apulmonary infection with one of the compositions or systems providedherein. In a further embodiment, the pulmonary infection is caused byMycobacterium abscessus or Mycobacterium avium complex.

In one embodiment, the concentration of the aminoglycoside in theliposomal aminoglycoside composition is about 50 mg/mL or greater. In afurther embodiment, the concentration of the aminoglycoside in theliposomal complexed aminoglycoside is about 60 mg/mL or greater. In afurther embodiment, the concentration of the aminoglycoside in theliposomal complexed aminoglycoside is about 70 mg/mL or greater, forexample about 70 mg/mL to about 75 mg/mL. In a further embodiment, theaminoglycoside is selected from an aminoglycoside provided in Table 1.In even a further embodiment, the aminoglycoside is amikacin (e.g.,amikacin sulfate).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the study design for a randomized, double-blind, placebocontrolled study of liposomal complexed amikacin in patients withrecalcitrant nontuberculous mycobacterial (NTM) lung infection,described in Example 1.

FIG. 2 shows the patient distribution for the randomized, double-blind,placebo controlled study of liposomal complexed amikacin in patientswith recalcitrant nontuberculous mycobacterial lung infection, describedin Example 1.

FIG. 3 shows the number of patients in each NTM treatment group.

FIG. 4 shows the log scale (LS) mean change from baseline on the fullsemi quantitative scale for mycobacterial culture for the modifiedintent to treat patient (mITT) population as a function of study day forboth the double-blind phase and the open-label phase of the study setforth in Example 1.

FIG. 5 (top) is a bar graph showing the proportion of patients with NTMculture conversion to negative at various time points during therandomized, double-blind, placebo controlled study (modified intent totreat population). FIG. 5 (bottom) is a bar graph showing the proportionof MAC patients with NTM culture conversion to negative at various timepoints.

FIG. 6 shows patients with at least 1 NTM culture negative result atvarious time points during the randomized, double-blind, placebocontrolled study.

FIG. 7 (top) is a graph showing the change from baseline in thesix-minute walk test at day 84 and day 168 (mITT population) and FIG. 7(bottom) is a graph of the mean change from baseline in distance walked(meters) in the 6MWT in patients receiving LAI vs. placebo at day 84(last observation carried forward, modified intent to treat population).

FIG. 8 (top) is a graph showing the average meters walked in thesix-minute walk test at day 84 and day 168 (all patients). FIG. 8(bottom) is a graph showing the mean change from baseline to Days 84 and168 in distance walked (meters) in the 6MWT in patients with cultureconversion to negative (≥3 negative cultures) vs. those without cultureconversion to negative (last observation carried forward-modified intentto treat population).

FIG. 9 shows the study design for a randomized, placebo controlled studyof liposomal encapsulated amikacin (ARIKAYCE or LAI) in patients withNon-Cystic Fibrosis (Non-CF) M. avium complex (MAC) lung infection,described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein is directed, in part, to methods fortreating a pulmonary infection in a patient in need thereof, e.g.,administering an aminoglycoside pharmaceutical composition to the lungsof the patient, for example, via nebulization.

The term “about,” as used herein, refers to plus or minus ten percent ofthe object that “about” modifies.

The term “treating” includes: (1) preventing or delaying the appearanceof clinical symptoms of the state, disorder or condition developing inthe subject that may be afflicted with or predisposed to the state,disorder or condition but does not yet experience or display clinical orsubclinical symptoms of the state, disorder or condition; (2) inhibitingthe state, disorder or condition (i.e., arresting, reducing or delayingthe development of the disease, or a relapse thereof in case ofmaintenance treatment, of at least one clinical or subclinical symptomthereof); and/or (3) relieving the condition (i.e., causing regressionof the state, disorder or condition or at least one of its clinical orsubclinical symptoms). The benefit to a subject to be treated is eitherstatistically significant or at least perceptible to the subject or tothe physician.

“Prophylaxis,” as used herein, can mean complete prevention of aninfection or disease, or prevention of the development of symptoms ofthat infection or disease; a delay in the onset of an infection ordisease or its symptoms; or a decrease in the severity of a subsequentlydeveloped infection or disease or its symptoms.

The term “antibacterial” is art-recognized and refers to the ability ofthe compounds of the present invention to prevent, inhibit or destroythe growth of microbes of bacteria. Examples of bacteria are providedabove.

The term “antimicrobial” is art-recognized and refers to the ability ofthe aminoglycoside compounds of the present invention to prevent,inhibit, delay or destroy the growth of microbes such as bacteria,fungi, protozoa and viruses.

“Effective amount” means an amount of an aminoglycoside (e.g., amikacin)used in the present invention sufficient to result in the desiredtherapeutic response. The effective amount of the composition providedherein comprises both free and liposomal complexed aminoglycoside. Forexample, the liposomal complexed aminoglycoside, in one embodiment,comprises aminoglycoside encapsulated in a liposome, or complexed with aliposome, or a combination thereof.

“Liposomal dispersion” refers to a solution or suspension comprising aplurality of liposomes.

An “aerosol,” as used herein, is a gaseous suspension of liquidparticles. The aerosol provided herein comprises particles of theliposomal dispersion.

A “nebulizer” or an “aerosol generator” is a device that converts aliquid into an aerosol of a size that can be inhaled into therespiratory tract. Pneumonic, ultrasonic, electronic nebulizers, e.g.,passive electronic mesh nebulizers, active electronic mesh nebulizersand vibrating mesh nebulizers are amenable for use with the invention ifthe particular nebulizer emits an aerosol with the required properties,and at the required output rate.

The process of pneumatically converting a bulk liquid into smalldroplets is called atomization. The operation of a pneumatic nebulizerrequires a pressurized gas supply as the driving force for liquidatomization. Ultrasonic nebulizers use electricity introduced by apiezoelectric element in the liquid reservoir to convert a liquid intorespirable droplets. Various types of nebulizers are described inRespiratory Care, Vol. 45, No. 6, pp. 609-622 (2000), the disclosure ofwhich is incorporated herein by reference in its entirety. The terms“nebulizer” and “aerosol generator” are used interchangeably throughoutthe specification. “Inhalation device,” “inhalation system” and“atomizer” are also used in the literature interchangeably with theterms “nebulizer” and “aerosol generator.”

“Mass median diameter” or “MMD” is determined by laser diffraction orimpactor measurements, and is the average particle diameter by mass.

“Mass median aerodynamic diameter” or “MMAD” is normalized regarding theaerodynamic separation of aqua aerosol droplets and is determinedimpactor measurements, e.g., the Anderson Cascade Impactor (ACI) or theNext Generation Impactor (NGI). The gas flow rate, in one embodiment, is28 Liter per minute by the Anderson Cascade Impactor (ACI) and 15 Literper minute by the Next Generation Impactor (NGI). “Geometric standarddeviation” or “GSD” is a measure of the spread of an aerodynamicparticle size distribution.

Nontuberculous mycobacteria are organisms found in the soil and waterthat can cause serious lung disease in susceptible individuals, forwhich there are currently limited effective treatments and no approvedtherapies. The prevalence of NTM disease is reported to be increasing,and according to reports from the American Thoracic Society is believedto be greater than that of tuberculosis in the U.S. According to theNational Center for Biotechnology Information, epidemiological studiesshow that presence of NTM infection is increasing in developingcountries, perhaps because of the implementation of tap water. Womenwith characteristic phenotype are believed to be at higher risk ofacquiring NTM infection along with patients with defects on cysticfibrosis transmembrane conductance regulators. Generally, high riskgroups with NTM lung disease for increased morbidity and mortality arethose with cavitary lesions, low BMI, advanced age, and a highcomorbidity index.

NTM lung disease is often a chronic condition that can lead toprogressive inflammation and lung damage, and is characterized bybronchiectasis and cavitary disease. NTM infections often requirelengthy hospital stays for medical management. Treatment usuallyinvolves multi-drug regimens that can be poorly tolerated and havelimited effectiveness, especially in patients with severe disease or inthose who have failed prior treatment attempts. According to acompany-sponsored patient chart study conducted by Clarity PharmaResearch, approximately 50,000 patients suffering from NTM lung diseasevisited physician offices in the U.S. during 2011.

Management of pulmonary disease caused by nontuberculous mycobacteria(NTM) infection includes lengthy multidrug regimens, which are oftenassociated with drug toxicity and suboptimal outcomes. Achieving NTMculture negativity is one of the objectives of treatment and representsthe most clinically important microbiologic endpoint in patients withNTM lung infection.

In one aspect, the present invention provides methods for treating apulmonary nontuberculous mycobacterial (NTM) infection in a patient inneed thereof. The method in one embodiment comprises administration tothe patient a composition comprising a liposomal complexedaminoglycoside, or a pharmaceutically acceptable salt thereof for anadministration period. The liposomal complexed aminoglycoside, in oneembodiment, comprises the aminoglycoside or pharmaceutically acceptablesalt thereof encapsulated in a plurality of liposomes. The plurality ofliposomes in one embodiment, include a lipid component that consists ofneutral lipids. In one embodiment, the neutral lipids comprise aphospholipid and a sterol. In a further embodiment, the phospholipid isa phosphatidylcholine. In even a further embodiment, thephosphatidylcholine is dipalmitoylphosphatidylcholine (DPPC). In even afurther embodiment, the sterol is cholesterol. In one embodiment, thenontuberculous mycobacterial lung infection is a recalcitrantnontuberculous mycobacterial lung infection. The patient, in oneembodiment, exhibits an increased number of meters walked in the 6MWT,as compared to prior to treatment and/or an NTM culture conversion tonegative, during the administration period or after the administrationperiod.

The therapeutic response can be any response that a user (e.g., aclinician) will recognize as an effective response to the therapy. Thetherapeutic response will generally be a reduction, inhibition, delay orprevention in growth of or reproduction of one or more NTM, or thekilling of one or more NTM. A therapeutic response may also be reflectedin an improvement in pulmonary function, for example forced expiratoryvolume in one second (FEV₁). In one embodiment, where a patient istreated for an NTM lung infection, the therapeutic response is measuredas the change from baseline on the full semi quantitative scale formycobacterial culture or an improvement in the distance walked in the 6minute walk test (6MWT). It is further within the skill of one ofordinary skill in the art to determine appropriate treatment duration,appropriate doses, and any potential combination treatments, based uponan evaluation of therapeutic response.

The NTM lung infection treatable by the methods and compositionsdescribed herein, in one embodiment, is M. avium, M. avium subsp.hominissuis (MAH), M. abscessus, M. chelonae, M. bolletii, M. kansasii,M. ulcerans, M. avium, M. avium complex (MAC) (M. avium and M.intracellulare), M. conspicuum, M. kansasii, M. peregrinum, M.immunogenum, M. xenopi, M. marinum, M. malmoense, M. marinum, M.mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. simiae, M.smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M.genavense, M. asiaticum, M. shimoidei, M. gordonae, M. nonchromogenicum,M. triplex, M. lentiflavum, M. celatum, M. fortuitum, M. fortuitumcomplex (M. fortuitum and M. chelonae) or a combination thereof. In afurther embodiment, the nontuberculous mycobacterial lung infection isM. avium complex (MAC) (M. avium and M. intracellulare), M. abscessus orM. avium. In a further embodiment, the M. avium infection is M. aviumsubsp. hominissuis. In one embodiment, the nontuberculous mycobacteriallung infection is M. avium complex (MAC) (M. avium and M.intracellulare). In another embodiment, the NTM lung infection is arecalcitrant nontuberculous mycobacterial lung infection.

As described throughout, the compositions and systems described hereinare used to treat an infection caused by a nontuberculous mycobacterium(NTM). In one embodiment, the compositions and systems described hereinare used to treat an infection caused by Mycobacterium abscessus,Mycobacterium avium or M. avium complex. In even a further embodiment,the Mycobacterium avium infection is Mycobacterium avium subsp.hominissuis.

In one embodiment, a patient is treated for a Mycobacterium abscessus,M. kansasii, M. abscessus, M. fortuitum, Mycobacterium avium or a M.avium complex (MAC) lung infection via inhalation delivery of aliposomal aminoglycoside composition. In a further embodiment, theaminoglycoside is amikacin sulfate and is administered once per day forin a single dosing session. In even a further embodiment, the NTM lunginfection is MAC.

The NTM lung infection, in one embodiment, is associated with cavitarylesions. In one embodiment, the NTM lung infection is a nodularinfection. In a further embodiment, the NTM lung infection is a nodularinfection with minimal cavitary lesions.

In one embodiment, the aminoglycoside or pharmaceutically acceptablesalt thereof, administered via the methods described herein, is selectedfrom amikacin, apramycin, arbekacin, astromicin, capreomycin, dibekacin,framycetin, gentamicin, hygromycin B, isepamicin, kanamycin, neomycin,netilmicin, paromomycin, rhodestreptomycin, ribostamycin, sisomicin,spectinomycin, streptomycin, tobramycin, verdamicin, or apharmaceutically acceptable salt thereof. In a further embodiment, theaminoglycoside is amikacin. In even a further embodiment, the amikacinis amikacin sulfate. In another embodiment, the aminoglycoside isselected from an aminoglycoside set forth in Table 2, below, apharmaceutically acceptable salt thereof, or a combination thereof. Forexample, a a pharmaceutically acceptable salt such as a sulfate salt ofone or more of the aminoglycosides set forth in Table 2 can beformulated in a liposomal composition and administered to a patient inneed of NTM treatment, e.g., via pulmonary delivery by a nebulizer.

TABLE 2 Aminoglycosides for use with the present invention AC4437dibekacin K-4619 sisomicin amikacin dactimicin isepamicinrhodestreptomycin arbekacin etimicin KA-5685 sorbistin apramycinframycetin kanamycin spectinomycin astromicin gentamicin neomycinsporaricin bekanamycin H107 netilmicin streptomycin boholmycinhygromycin paromomycin tobramycin brulamycin hygromycin B plazomicinverdamicin capreomycin inosamycin ribostamycin vertilmicin

In one embodiment, a pharmaceutical composition comprises a combinationof aminoglycosides, or pharmaceutically acceptable salts thereof, e.g.,a combination of two or more aminoglycosides, or pharmaceuticallyacceptable salts thereof, as set forth in Table 2. In one embodiment,the composition comprising the liposomal complexed aminoglycosidecomprises from 1 to about 5 aminoglycosides, or pharmaceuticallyacceptable salts thereof. In another embodiment, the compositioncomprising the liposomal complexed aminoglycoside comprises at least 1,at least 2, at least 3, at least 4, at least 5, or at least 6, of theaminoglycosides set forth in table 2 (or pharmaceutically acceptablesalts of the aminoglycosides. In another embodiment, a pharmaceuticalcomposition comprises between 1 and 4 aminoglycosides, orpharmaceutically acceptable salts thereof. In a further embodiment, thecombination comprises amikacin, e.g., as amikacin sulfate.

In one embodiment, the aminoglycoside is an aminoglycoside free base, orits salt, solvate, or other non-covalent derivative. In a furtherembodiment, the aminoglycoside is amikacin. Included as suitableaminoglycosides used in the drug compositions of the present inventionare pharmaceutically acceptable addition salts and complexes of drugs.In cases where the compounds may have one or more chiral centers, unlessspecified, the present invention comprises each unique racemic compound,as well as each unique nonracemic compound. In cases in which the activeagents have unsaturated carbon-carbon double bonds, both the cis (Z) andtrans (E) isomers are within the scope of this invention. In cases wherethe active agents exist in tautomeric forms, such as keto-enoltautomers, each tautomeric form is contemplated as being included withinthe invention. Amikacin, in one embodiment, is present in thepharmaceutical composition as amikacin base, or amikacin salt, forexample, amikacin sulfate or amikacin disulfate. In one embodiment, acombination of one or more of the above aminoglycosides is used in thecompositions, systems and methods described herein.

The present invention provides in one aspect, a method for treating orproviding prophylaxis against a pulmonary NTM infection. Treatment isachieved via delivery of a composition comprising a liposomalaminoglycoside composition by inhalation via nebulization of thecomposition. In one embodiment, the composition comprises anaminoglycoside encapsulated in a plurality of liposomes, e.g., anaminoglycoside selected from one or more of the aminoglycosides ofTables 1 and/or 2, or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition, as provided herein, is a liposomaldispersion comprising an aminoglycoside complexed to a liposome, e.g.,an aminoglycoside encapsulated in a plurality of liposomes. Thepharmaceutical composition is a dispersion comprising a “liposomalcomplexed aminoglycoside” or an “aminoglycoside encapsulated in aliposome.” A “liposomal complexed aminoglycoside” includes embodimentswhere the aminoglycoside (or combination of aminoglycosides) isencapsulated in a liposome, and includes any form of aminoglycosidecomposition where at least about 1% by weight of the aminoglycoside isassociated with the liposome either as part of a complex with aliposome, or as a liposome where the aminoglycoside may be in theaqueous phase or the hydrophobic bilayer phase or at the interfacialheadgroup region of the liposomal bilayer.

In one embodiment, the lipid component of the liposome or plurality ofliposomes comprises electrically neutral lipids, positively chargedlipids, negatively charged lipids, or a combination thereof. In anotherembodiment, the lipid component comprises electrically neutral lipids.In a further embodiment, the lipid component consists essentially ofelectrically neutral lipids. In even a further embodiment, theelectrically neutral lipids comprise a sterol and a phospholipid. Ineven a further embodiment the sterol is cholesterol and the phospholipidis a neutral phosphatidylcholine. In one embodiment, thephosphatidylcholine is dipalmitoylphosphatidylcholine (DPPC).

As provided above, liposomal complexed aminoglycoside embodimentsinclude embodiments where the aminoglycoside or pharmaceuticallyacceptable salt thereof is encapsulated in a plurality of liposomes. Inaddition, the liposomal complexed aminoglycoside describes anycomposition, solution or suspension where at least about 1% by weight ofthe aminoglycoside is associated with the lipid either as part of acomplex with the liposome, or as a liposome where the aminoglycoside maybe in the aqueous phase or the hydrophobic bilayer phase or at theinterfacial headgroup region of the liposomal bilayer. In oneembodiment, prior to nebulization, at least about 5%, at least about10%, at least about 20%, at least about 25%, at least about 50%, atleast about 75%, at least about 80%, at least about 85%, at least about90% or at least about 95% of the aminoglycoside in the composition is soassociated. Association, in one embodiment, is measured by separationthrough a filter where lipid and lipid-associated drug is retained(i.e., in the retentate) and free drug is in the filtrate.

The methods provided herein comprise administering to a patient in needthereof a composition comprising an aminoglycoside or pharmaceuticallyacceptable salt thereof encapsulated in a plurality of liposomes. One ormore lipids can be used to form the plurality of liposomes. In oneembodiment, the one or more lipids is synthetic, semi-synthetic or anaturally-occurring lipid, including a phospholipid, tocopherol, sterol,fatty acid, negatively-charged lipid, cationic lipid or a combinationthereof. In one embodiment, the lipid component of the plurality ofliposomes consists of electrically neutral lipids. In a furtherembodiment, the lipid component comprises DPPC and cholesterol.

In one embodiment, at least one phospholipid is present in the pluralityof liposomes. The phospholipid, in one embodiment, is electrically netneutral. In one embodiment, the phospholipid is a phosphatidylcholine(PC), phosphatidylglycerol (PG), phosphatidylinositol (PI),phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidicacid (PA); the soya counterparts, soy phosphatidylcholine (SPC); SPG,SPS, SPI, SPE, and SPA; the hydrogenated egg and soya counterparts(e.g., HEPC, HSPC), phospholipids made up of ester linkages of fattyacids in the 2 and 3 of glycerol positions containing chains of 12 to 26carbon atoms and different head groups in the 1 position of glycerolthat include choline, glycerol, inositol, serine, ethanolamine, as wellas the corresponding phosphatidic acids. The carbon chains on thesefatty acids can be saturated or unsaturated, and the phospholipid may bemade up of fatty acids of different chain lengths and different degreesof unsaturation.

In one embodiment, the lipid component of the plurality of liposomesincludes dipalmitoylphosphatidylcholine (DPPC), a major constituent ofnaturally-occurring lung surfactant. In one embodiment, the lipidcomponent of the plurality of liposomes comprises DPPC and cholesterol,or consists essentially of DPPC and cholesterol, or consists of DPPC andcholesterol. In a further embodiment, the DPPC and cholesterol have amole ratio in the range of from about 19:1 to about 1:1, or about 9:1 toabout 1:1, or about 4:1 to about 1:1, or about 2:1 to about 1:1, orabout 1.86:1 to about 1:1. In even a further embodiment, the DPPC andcholesterol have a mole ratio of about 2:1 or about 1:1.

Other examples of lipids for use with the methods and compositionsdescribed herein include, but are not limited to,dimyristoylphosphatidycholine (DMPC), dimyri stoylphosphatidylglycerol(DMPG), dipalmitoylphosphatidcholine (DPPC),dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine(DSPC), distearoylphosphatidylglycerol (DSPG),dioleylphosphatidyl-ethanolamine (DOPE), mixed phospholipids such aspalmitoylstearoylphosphatidyl-choline (PSPC), and single acylatedphospholipids, for example, mono-oleoyl-phosphatidylethanolamine (MOPE).

In one embodiment, the lipid component of the plurality of liposomescomprises a sterol. In a further embodiment, the at least one lipidcomponent comprises a sterol and a phospholipid, or consists essentiallyof a sterol and a phospholipid, or consists of a sterol and aphospholipid (e.g., a neutral phosphatidylcholine such as DPPC). Sterolsfor use with the invention include, but are not limited to, cholesterol,esters of cholesterol including cholesterol hemi-succinate, salts ofcholesterol including cholesterol hydrogen sulfate and cholesterolsulfate, ergosterol, esters of ergosterol including ergosterolhemi-succinate, salts of ergosterol including ergosterol hydrogensulfate and ergosterol sulfate, lanosterol, esters of lanosterolincluding lanosterol hemi-succinate, salts of lanosterol includinglanosterol hydrogen sulfate, lanosterol sulfate and tocopherols. Thetocopherols can include tocopherols, esters of tocopherols includingtocopherol hemi-succinates, salts of tocopherols including tocopherolhydrogen sulfates and tocopherol sulfates. The term “sterol compound”includes sterols, tocopherols and the like.

In one embodiment, at least one cationic lipid (positively chargedlipid) is provided in the lipid component of the plurality of liposomes,present in the liposomal aminoglycoside compositions described herein,for use in the method of treating an NTM pulmonary infection in apatient in need thereof. Cationic lipids amendable for use with thepresent invention include but are not limited to ammonium salts of fattyacids, phospholids and glycerides. The fatty acids include fatty acidsof carbon chain lengths of 12 to 26 carbon atoms that are eithersaturated or unsaturated. Some specific examples include, but are notlimited to, myristylamine, palmitylamine, laurylamine and stearylamine,dilauroyl ethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine(DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoylethylphosphocholine (DSEP),N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio) propane(DOTAP), and combinations thereof.

In one embodiment, at least one anionic lipid (negatively charged lipid)is provided in the lipid component of the plurality of liposomes,present in the liposomal aminoglycoside compositions described herein,for use in the method of treating an NTM pulmonary infection in apatient in need thereof. The negatively-charged lipids which can be usedinclude phosphatidyl-glycerols (PGs), phosphatidic acids (PAs),phosphatidylinositols (PIs) and the phosphatidyl serines (PSs). Examplesinclude but are not limited to DMPG, DPPG, DSPG, DMPA, DPPA, DSPA, DMPI,DPPI, DSPI, DMPS, DPPS, DSPS and combinations thereof.

Without wishing to be bound by theory, phosphatidylcholines, such asDPPC, aid in the uptake of the aminoglycoside agent by the cells in thelung (e.g., the alveolar macrophages) and helps to maintain theaminoglycoside agent in the lung. The negatively charged lipids such asthe PGs, PAs, PSs and PIs, in addition to reducing particle aggregation,are thought to play a role in the sustained activity characteristics ofthe inhalation composition as well as in the transport of thecomposition across the lung (transcytosis) for systemic uptake. Thesterol compounds, without wishing to be bound by theory, are thought toaffect the release characteristics of the composition.

Liposomes are completely closed lipid bilayer membranes containing anentrapped aqueous volume. Liposomes may be unilamellar vesicles(possessing a single membrane bilayer) or multilamellar vesicles(onion-like structures characterized by multiple membrane bilayers, eachseparated from the next by an aqueous layer) or a combination thereof.The bilayer is composed of two lipid monolayers having a hydrophobic“tail” region and a hydrophilic “head” region. The structure of themembrane bilayer is such that the hydrophobic (nonpolar) “tails” of thelipid monolayers orient toward the center of the bilayer while thehydrophilic “heads” orient towards the aqueous phase.

The lipid to aminoglycoside ratio by weight (weight ratios are alsoreferred to herein as “lipid:aminoglycoside”) in the pharmaceuticalcomposition provided herein, in one embodiment, is 3:1 or less, 2.5:1.0or less, 2:1 or less, 1.5:1 or less, 1:1 or less or 0.75:1 or less. Inone embodiment, the lipid:aminoglycoside weight ratio in the compositionprovided herein is 0.7:1.0 or about 0.7:1.0 by weight. In anotherembodiment, the L:D ratio in liposomes provided herein is 0.75:1 or less(by weight). In one embodiment, the lipid:aminoglycoside weight ratio(lipid to aminoglycoside weight ratio) is from about 0.10:1.0 to about1.25:1.0, from about 0.25:1.0 to about 1.25:1.0, from about 0.50:1.0 toabout 1.25:1.0 or from about 0.6:1 to about 1.25:1.0. In anotherembodiment, the lipid to aminoglycoside weight ratio is from about0.1:1.0 to about 1.0:1.0, or from about 0.25:1.0 to about 1.0:1.0 orabout 0.5:1 to 1:1.0.

The lipid to aminoglycoside weight ratio in the composition providedherein in another embodiment, is less than 3:1, less than 2.5:1.0, lessthan 2.0:1.0, less than 1.5:1.0, or less than 1.0:1.0. In a furtherembodiment, the lipid to aminoglycoside weight ratio is about 0.7:1.0 orless or about 0.7:1.0. In yet another embodiment, the lipid toaminoglycoside weight ratio is from about 0.5:1.0 to about 0.8:1.0.

In order to minimize dose volume and reduce patient dosing time, in oneembodiment, it is important that liposomal entrapment of theaminoglycoside (e.g., the aminoglycoside amikacin) be highly efficientand that the lipid to aminoglycoside weight ratio be at as low a valueas possible and/or practical while keeping the liposomes small enough topenetrate patient mucus and biofilms. In one embodiment, the Laminoglycoside weight ratio in the composition provided herein, i.e.,the composition comprising an aminoglycoside encapsulated in a pluralityof liposomes is 0.7:1.0, about 0.7:1.0 from about 0.5:1.0 to about0.8:1.0 or from about 0.6:1.0 to about 0.8:1.0. In a further embodiment,the liposomes provided herein are small enough to effectively penetratea bacterial biofilm. In even a further embodiment, the mean diameter ofthe plurality of liposomes, as measured by light scattering is fromabout 200 nm to about 400 nm, or from about 250 nm to about 400 nm, orfrom about 250 nm to about 300 nm, or from about 200 nm to about 300 nm.In even a further embodiment, the mean diameter of the plurality ofliposomes, as measured by light scattering is from about 260 to about280 nm.

In one embodiment, the liposomal compositions described herein aremanufactured by one of the methods set forth in U.S. Patent ApplicationPublication No. 2013/0330400 or U.S. Pat. No. 7,718,189, each of whichis incorporated by reference in its entirety for all purposes. Liposomescan be produced by a variety of methods (see, e.g., Cullis et al.(1987)). In one embodiment, one or more of the methods described in U.S.Patent Application Publication No. 2008/0089927 are used herein toproduce the aminoglycoside encapsulated lipid compositions (liposomaldispersion). The disclosure of U.S. Patent Application Publication No.2008/0089927 is incorporated by reference in its entirety for allpurposes. For example, in one embodiment, at least one lipid and anaminoglycoside are mixed with a coacervate (i.e., a separate liquidphase) to form the liposome composition. The coacervate can be formed toprior to mixing with the lipid, during mixing with the lipid or aftermixing with the lipid. Additionally, the coacervate can be a coacervateof the active agent.

In one embodiment, the liposomal dispersion is formed by dissolving oneor more lipids in an organic solvent forming a lipid solution, and theaminoglycoside coacervate forms from mixing an aqueous solution of theaminoglycoside with the lipid solution. In a further embodiment, theorganic solvent is ethanol. In even a further embodiment, the lipidsolution comprises a phospholipid and a sterol, e.g., DPPC andcholesterol.

In one embodiment, liposomes are produces by sonication, extrusion,homogenization, swelling, electroformation, inverted emulsion or areverse evaporation method. Bangham's procedure (J. Mol. Biol. (1965))produces ordinary multilamellar vesicles (MLVs). Lenk et al. (U.S. Pat.Nos. 4,522,803, 5,030,453 and 5,169,637), Fountain et al. (U.S. Pat. No.4,588,578) and Cullis et al. (U.S. Pat. No. 4,975,282) disclose methodsfor producing multilamellar liposomes having substantially equalinterlamellar solute distribution in each of their aqueous compartments.Paphadjopoulos et al., U.S. Pat. No. 4,235,871, discloses preparation ofoligolamellar liposomes by reverse phase evaporation. Each of themethods is amenable for use with the present invention.

Unilamellar vesicles can be produced from MLVs by a number oftechniques, for example, the extrusion techniques of U.S. Pat. Nos.5,008,050 and 5,059,421. Sonication and homogenization cab be so used toproduce smaller unilamellar liposomes from larger liposomes (see, forexample, Paphadjopoulos et al. (1968); Deamer and Uster (1983); andChapman et al. (1968)).

The liposome preparation of Bangham et al. (J. Mol. Biol. 13, 1965, pp.238-252) involves suspending phospholipids in an organic solvent whichis then evaporated to dryness leaving a phospholipid film on thereaction vessel. Next, an appropriate amount of aqueous phase is added,the 60 mixture is allowed to “swell,” and the resulting liposomes whichconsist of multilamellar vesicles (MLVs) are dispersed by mechanicalmeans. This preparation provides the basis for the development of thesmall sonicated unilamellar vesicles described by Papahadjopoulos et al.(Biochim. Biophys. Acta. 135, 1967, pp. 624-638), and large unilamellarvesicles.

Techniques for producing large unilamellar vesicles (LUVs), such as,reverse phase evaporation, infusion procedures, and detergent dilution,can be used to produce liposomes for use in the pharmaceuticalcompositions provided herein. A review of these and other methods forproducing liposomes may be found in the text Liposomes, Marc Ostro, ed.,Marcel Dekker, Inc., New York, 1983, Chapter 1, which is incorporatedherein by reference. See also Szoka, Jr. et al., (Ann. Rev. Biophys.Bioeng. 9, 1980, p. 467), which is also incorporated herein by referencein its entirety for all purposes.

Other techniques for making liposomes include those that formreverse-phase evaporation vesicles (REV), U.S. Pat. No. 4,235,871.Another class of liposomes that may be used is characterized as havingsubstantially equal lamellar solute distribution. This class ofliposomes is denominated as stable plurilamellar vesicles (SPLV) asdefined in U.S. Pat. No. 4,522,803, and includes monophasic vesicles asdescribed in U.S. Pat. No. 4,588,578, and frozen and thawedmultilamellar vesicles (FATMLV) as described above.

A variety of sterols and their water soluble derivatives such ascholesterol hemisuccinate have been used to form liposomes; see, e.g.,U.S. Pat. No. 4,721,612. Mayhew et al., PCT Publication No. WO 85/00968,described a method for reducing the toxicity of drugs by encapsulatingthem in liposomes comprising alpha-tocopherol and certain derivativesthereof. Also, a variety of tocopherols and their water solublederivatives have been used to form liposomes, see PCT Publication No.87/02219.

The pharmaceutical composition, in one embodiment, pre-nebulization,comprises liposomes with a mean diameter, that is measured by a lightscattering method, of approximately 0.01 microns to approximately 3.0microns, for example, in the range about 0.2 to about 1.0 microns. Inone embodiment, the mean diameter of the liposomes in the composition isabout 200 nm to about 300 nm, about 210 nm to about 290 nm, about 220 nmto about 280 nm, about 230 nm to about 280 nm, about 240 nm to about 280nm, about 250 nm to about 280 nm or about 260 nm to about 280 nm. Thesustained activity profile of the liposomal product can be regulated bythe nature of the lipid membrane and by inclusion of other excipients inthe composition.

In one embodiment, the method described herein comprises administering aliposomal complexed aminoglycoside composition, e.g., a liposomalcomplexed amikacin (e.g., amikacin sulfate) composition to a patient inneed thereof via inhalation, for example, via a nebulizer. In oneembodiment, the amount of aminoglycoside provided in the composition isabout 450 mg, about 500 mg, about 550 mg, about 560 mg, about 570 mg,about 580 mg, about 590 mg, about 600 mg or about 610 mg. In anotherembodiment, the amount of aminoglycoside provided in the composition isfrom about 500 mg to about 600 mg, or from about 500 mg to about 650 mg,or from about 525 mg to about 625 mg, or from about 550 mg to about 600mg. In one embodiment, the amount of aminoglycoside administered to thesubject is about 560 mg and is provided in an 8 mL composition. In oneembodiment, the amount of aminoglycoside administered to the subject isabout 590 mg and is provided in an 8 mL composition. In one embodiment,the amount of aminoglycoside administered to the subject is about 600 mgand is provided in an 8 mL composition. In one embodiment, theaminoglycoside is amikacin and the amount of amikacin provided in thecomposition is about 450 mg, about 500 mg, about 550 mg, about 560 mg,about 570 mg, about 580 mg, about 590 mg, about 600 mg or about 610 mg.In another embodiment, the aminoglycoside is amikacin and the amount ofamikacin provided in the composition is from about 500 mg to about 650mg, or from about 525 mg to about 625 mg, or from about 550 mg to about600 mg. In one embodiment, the aminoglycoside is amikacin and the amountof amikacin administered to the subject is about 560 mg, and is providedin an 8 mL composition. In one embodiment, the aminoglycoside isamikacin and the amount of amikacin administered to the subject is about590 mg, and is provided in an 8 mL composition. In one embodiment, theaminoglycoside is amikacin and the amount of aminoglycoside administeredto the subject is about 600 mg and is provided in an 8 mL composition.

In one embodiment, the methods described herein are carried out via theuse of a system comprising a liposomal complexed aminoglycosidecomposition, for example, a liposomal encapsulated amikacin composition(e.g., amikacin sulfate) and a nebulizer. In one embodiment, theliposomal aminoglycoside composition provided herein comprises about 60mg/mL aminoglycoside, about 65 mg/mL aminoglycoside, about 70 mg/mLaminoglycoside, about 75 mg/mL aminoglycoside, about 80 mg/mLaminoglycoside, about 85 mg/mL aminoglycoside, or about 90 mg/mLaminoglycoside. In a further embodiment, the aminoglycoside is amikacin,for example, as amikacin sulfate.

In one embodiment of the NTM treatment methods described herein, theliposomal aminoglycoside composition is administered to a patient inneed thereof once per day in a single dosing session. In a furtherembodiment, the composition is administered as an aerosol via anebulizer. In another embodiment, the method comprises administering toa patient in need thereof one of the aminoglycoside compositionsdescribed herein every other day or every three days. In yet anotherembodiment, the method comprises administering to a patient in needthereof one of the aminoglycoside compositions described herein twiceper day.

The methods provided herein, in one embodiment, comprise administeringto a patient in need thereof one of the compositions described herein(e.g., via a nebulizer) for an administration period comprising at leastone 1 month, 2 months, 3 months, 4 months, 5 months or 6 months. In oneembodiment, an administration period is followed by a period where nocomposition is administered (referred to as “off period”), which isfollowed by another administration period. The off period, in oneembodiment is about 1 month, about 2 months, about 3 months, about fourmonths, about five months or about 6 months.

In one embodiment, the administration period is from about 15 days toabout 400 days, e.g., from about 45 days to about 300 days, or fromabout 45 days to about 270 days, or from about 80 days to about 200days. In one embodiment, the administration period comprisesadministration of the composition to a patient in need thereof in a oncedaily dosing session.

In another embodiment, the NTM treatment method described hereincomprises administration of a liposomal complexed aminoglycosidecomposition to a patient in need thereof via a once daily dosing sessionfor an administration period. In a further embodiment, theadministration period is from about 15 to about 275 days, or from about20 to about 235 days, or from about 28 days to about 150 days. Forexample, the methods provided herein comprise administering to a patientin need thereof an aminoglycoside composition once per day in a singledosing session for an administration period of from about 15 to about300 days, or from about 15 to about 250 days, or from about 15 to about200 days, or from about 15 to about 150 days, or from about 15 to about125 days or from about 15 to about 100 days. In another embodiment, theadministration period is from about 50 days to about 200 days. Duringthe administration period, in one embodiment, the patient in needthereof is administered the aminoglycoside composition via nebulization,and about 500 mg to about 1000 mg aminoglycoside is administered dailyin a single dosing session, for example, about 500 mg aminoglycoside toabout 700 mg aminoglycoside (e.g., about 590 mg aminoglycoside).

In one embodiment, an administration period is followed by an off periodfrom about 15 to about 200 days, for example, from about 15 days toabout 150 days, or from about 15 days to about 75 days, from about 15days to about 35 days, or from about 20 days to about 35 days, or fromabout 25 days to about 75 days, or from about 35 days to about 75 daysor from about 45 days to about 75 days. In another embodiment, the offperiod is about 28 days or about 56 days. In other embodiments, the offperiod is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days, whilein other embodiments, the off period is about 56 days.

In one embodiment, the patient in need thereof is administered theliposomal complexed aminoglycoside composition in a treatment cyclecomprising an administration period and an off period. In a furtherembodiment, the treatment cycle is implemented at least once. In afurther embodiment, the treatment cycle is repeated at least twice, forexample, two, three, four, five, six, seven, eight, nine or ten times.In another embodiment, the treatment cycle is repeated at least threetimes, for example, at least three, at least four, at least five or atleast six times.

Various treatment cycles for patients with NTM lung infections areprovided in Table 3, below. However, in another embodiment, the methodprovided herein does not comprise an off period and instead includesonly an administration period. In a further embodiment, one of theadministration periods set forth in Table 3 is used in the methodprovided herein. In a further embodiment, the patient is administeredthe liposomal aminoglycoside composition once daily during theadministration period in a single dosing session.

TABLE 3 Treatment cycles of the present invention AdministrationTreatment period Off period cycle(s) Composition 15 to 500 days 15 to 75days At least once Amikacin (500 mg-600 mg), DPPC, cholesterol, (lipidto aminoglycoside ratio by weight of 0.75:1 or less, e.g., 0.1:1.0 toabout 1.25:1.0) 15 to 450 days 15 to 75 days At least once Amikacin (500mg-600 mg), DPPC, cholesterol, (lipid to aminoglycoside ratio by weightof 0.75:1 or less, e.g., 0.1:1.0 to about 1.25:1.0) 15 to 400 days 15 to75 days At least once Amikacin (500 mg-600 mg), DPPC, cholesterol,(lipid to aminoglycoside ratio by weight of 0.75:1 or less, e.g.,0.1:1.0 to about 1.25:1.0) 15 to 350 days 15 to 75 days At least onceAmikacin (500 mg-600 mg), DPPC, cholesterol, (lipid to aminoglycosideratio by weight of 0.75:1 or less, e.g., 0.1:1.0 to about 1.25:1.0) 15to 325 days 15 to 75 days At least once Amikacin (500 mg-600 mg), DPPC,cholesterol, (lipid to aminoglycoside ratio by weight of 0.75:1 or less,e.g., 0.1:1.0 to about 1.25:1.0) 15 to 300 days 15 to 75 days At leastonce Amikacin (500 mg-600 mg), DPPC, cholesterol, (lipid toaminoglycoside ratio by weight of 0.75:1 or less, e.g., 0.1:1.0 to about1.25:1.0) 15 to 275 days 15 to 75 days At least once Amikacin (500mg-600 mg), DPPC, cholesterol, (lipid to aminoglycoside ratio by weightof 0.75:1 or less, e.g., 0.1:1.0 to about 1.25:1.0) 15 to 255 days 15 to75 days At least once Amikacin (500 mg-600 mg), DPPC, cholesterol,(lipid to aminoglycoside ratio by weight of 0.75:1 or less, e.g.,0.1:1.0 to about 1.25:1.0) 15 to 225 days 15 to 75 days At least onceAmikacin (500 mg-600 mg), DPPC, cholesterol, (lipid to aminoglycosideratio by weight of 0.75:1 or less, e.g., 0.1:1.0 to about 1.25:1.0) 15to 200 days 15 to 75 days At least once Amikacin (500 mg-600 mg), DPPC,cholesterol, (lipid to aminoglycoside ratio by weight of 0.75:1 or less,e.g., 0.1:1.0 to about 1.25:1.0) 15 to 175 days 15 to 75 days At leastonce Amikacin (500 mg-600 mg), DPPC, cholesterol, (lipid toaminoglycoside ratio by weight of 0.75:1 or less, e.g., 0.1:1.0 to about1.25:1.0) 15 to 150 days 15 to 75 days At least once Amikacin (500mg-600 mg), DPPC, cholesterol, (lipid to aminoglycoside ratio by weightof 0.75:1 or less, e.g., 0.1:1.0 to about 1.25:1.0) 15 to 125 days 15 to75 days At least once Amikacin (500 mg-600 mg), DPPC, cholesterol,(lipid to aminoglycoside ratio by weight of 0.75:1 or less, e.g.,0.1:1.0 to about 1.25:1.0) 15 to 100 days 15 to 75 days At least onceAmikacin (about 590 mg), DPPC, cholesterol, (L:D by weight of about0.7:1) 15 to 75 days 15 to 75 days At least once Amikacin (about 590mg), DPPC, cholesterol, (L:D by weight of about 0.7:1) 15 to 50 days 15to 75 days At least once Amikacin (about 590 mg), DPPC, cholesterol,(L:D by weight of about 0.7:1) 20 to 100 days 15 to 75 days At leastonce Amikacin (about 590 mg), DPPC, cholesterol, (L:D by weight of about0.7:1)

In one embodiment, the system provided herein comprises an about 8 mLliposomal amikacin composition and a nebulizer. In one embodiment, thedensity of the liposomal amikacin composition is about 1.05 gram/mL; andin one embodiment, approximately 8.4 grams of the liposomal amikacincomposition per dose is present in the composition of the invention. Ina further embodiment, the entire volume of the composition isadministered to a subject in need thereof.

In one embodiment, the pharmaceutical composition provided hereincomprises at least one aminoglycoside, at least one phospholipid and asterol. In a further embodiment, the pharmaceutical compositioncomprises an aminoglycoside, DPPC and cholesterol. In one embodiment,the pharmaceutical composition is the composition provided in Table 4,below.

TABLE 4 Pharmaceutical Compositions Component Concentration CompositionA (pH 6.0-7.0) Aminoglycoside 60-80 mg/mL Phospholipid 30-40 mg/mLSterol 10-20 mg/mL Salt 0.5%-5.0% Composition B (pH 6.0-7.0) AmikacinSulfate 60-80 mg/mL DPPC 30-40 mg/mL Cholesterol 10-20 mg/mL NaCl0.5%-5.0% Composition C (pH 6.0-7.0) Amikacin Sulfate 70-80 mg/mL DPPC35-40 mg/mL Cholesterol 15-20 mg/mL NaCl 0.5%-5.0% Composition D (pH~6.5) Aminoglycoside ~70 mg/mL Phospholipid ~32-35 mg/mL Sterol ~16-17mg/mL Salt ~1.5% Composition E (pH ~6.5) Amikacin Sulfate ~70 mg/mL DPPC~32-35 mg/mL Cholesterol ~16-17 mg/mL NaCl ~1.5% Composition F (pH ~6.5)Amikacin Sulfate ~70 mg/mL DPPC ~30-35 mg/mL Cholesterol ~15-17 mg/mLNaCl ~1.5%

It should be noted that increasing aminoglycoside concentration alonemay not result in a reduced dosing time. For example, in one embodiment,the lipid to drug ratio is fixed, and as amikacin concentration isincreased (and therefore lipid concentration is increased, since theratio of the two is fixed, for example at ˜0.7:1 by weight), theviscosity of the solution also increases, which slows nebulization time.

As provided throughout, the methods described herein compriseadministering to a patient in need of treatment of an NTM lunginfection, an effective amount of a liposomal aminoglycoside compositionvia inhalation. In one embodiment, inhalation delivery is conducted viaa nebulizer. The nebulizer provides an aerosol mist of the compositionfor delivery to the lungs of the patient.

In one embodiment, the system provided herein comprises a nebulizerselected from an electronic mesh nebulizer, pneumonic (jet) nebulizer,ultrasonic nebulizer, breath-enhanced nebulizer and breath-actuatednebulizer. In one embodiment, the nebulizer is portable.

In one embodiment, the method for treating an NTM infection is carriedout via administration of a liposomal complexed aminoglycosidecomposition to a patient in need thereof via a nebulizer in once dailydosing sessions. In a further embodiment, the aminoglycoside isamikacin, e.g., amikacin sulfate. In a further embodiment, the lipidcomponent of the liposomes comprises DPPC and cholesterol. In even afurther embodiment, the nebulizer is one of the nebulizers described inU.S. Patent Application Publication No. 2013/0330400, incorporated byreference herein in its entirety for all purposes.

The principle of operation of a pneumonic nebulizer is generally knownto those of ordinary skill in the art and is described, e.g., inRespiratory Care, Vol. 45, No. 6, pp. 609-622 (2000). Briefly, apressurized gas supply is used as the driving force for liquidatomization in a pneumatic nebulizer. Compressed gas is delivered, whichcauses a region of negative pressure. The solution to be aerosolized isthen delivered into the gas stream and is sheared into a liquid film.This film is unstable and breaks into droplets because of surfacetension forces. Smaller particles, i.e., particles with the MMAD and FPFproperties described above, can then be formed by placing a baffle inthe aerosol stream. In one pneumonic nebulizer embodiment, gas andsolution is mixed prior to leaving the exit port (nozzle) andinteracting with the baffle. In another embodiment, mixing does not takeplace until the liquid and gas leave the exit port (nozzle). In oneembodiment, the gas is air, O₂ and/or CO₂.

In one embodiment, droplet size and output rate can be tailored in apneumonic nebulizer. However, consideration should be paid to thecomposition being nebulized, and whether the properties of thecomposition (e.g., % associated aminoglycoside) are altered due to themodification of the nebulizer. For example, in one embodiment, the gasvelocity and/or pharmaceutical composition velocity is modified toachieve the output rate and droplet sizes of the present invention.Additionally or alternatively, the flow rate of the gas and/or solutioncan be tailored to achieve the droplet size and output rate of theinvention. For example, an increase in gas velocity, in one embodiment,decreased droplet size. In one embodiment, the ratio of pharmaceuticalcomposition flow to gas flow is tailored to achieve the droplet size andoutput rate of the invention. In one embodiment, an increase in theratio of liquid to gas flow increases particle size.

In one embodiment, a pneumonic nebulizer output rate is increased byincreasing the fill volume in the liquid reservoir. Without wishing tobe bound by theory, the increase in output rate may be due to areduction of dead volume in the nebulizer. Nebulization time, in oneembodiment, is reduced by increasing the flow to power the nebulizer.See, e.g., Clay et al. (1983). Lancet 2, pp. 592-594 and Hess et al.(1996). Chest 110, pp. 498-505.

In one embodiment, a reservoir bag is used to capture aerosol during thenebulization process, and the aerosol is subsequently provided to thesubject via inhalation. In another embodiment, the nebulizer providedherein includes a valved open-vent design. In this embodiment, when thepatient inhales through the nebulizer, nebulizer output is increased.During the expiratory phase, a one-way valve diverts patient flow awayfrom the nebulizer chamber.

In one embodiment, the nebulizer provided herein is a continuousnebulizer. In other words, refilling the nebulizer with thepharmaceutical composition while administering a dose is not needed.Rather, the nebulizer has at least an 8 mL capacity or at least a 10 mLcapacity.

In one embodiment, the nebulizer provided herein does not use an aircompressor and therefore does not generate an air flow. In oneembodiment, aerosol is produced by the aerosol head which enters themixing chamber of the device. When the patient inhales, air enters themixing chamber via one-way inhalation valves in the back of the mixingchamber and carries the aerosol through the mouthpiece to the patient.On exhalation, the patient's breath flows through the one-way exhalationvalve on the mouthpiece of the device. In one embodiment, the nebulizercontinues to generate aerosol into the mixing chamber which is thendrawn in by the subject on the next breath—and this cycle continuesuntil the nebulizer medication reservoir is empty.

In one embodiment, the nebulization time of an effective amount of anaminoglycoside composition provided herein is less than 20 minutes, lessthan 18 minutes, less than 16 minutes or less than 15 minutes. In oneembodiment, the nebulization time of an effective amount of anaminoglycoside composition provided herein is less than 15 minutes orless than 13 minutes. In one embodiment, the nebulization time of aneffective amount of an aminoglycoside composition provided herein isabout 13 minutes.

In one embodiment, the composition described herein is administered oncedaily to a patient in need thereof.

In another embodiment, a patient is treated for an NTM lung infectionwith one of the methods and/or compositions provided herein. In afurther embodiment, the composition comprises a liposomal amikacincomposition. In even a further embodiment, the composition comprisesfrom about 500 mg to about 600 mg amikacin, DPPC and cholesterol, andthe lipid to aminoglycoside weight ratio of the composition is 0.75:1.0or less, e.g., about 0.7:1.0 or about 0.5:1.0 to about 0.8:1.0.

In one embodiment, the patient subjected to one of the treatment methodsprovided herein is a patient that was previously non-responsive to adifferent NTM treatment. In a further embodiment, the compositionadministered to the patient in need of treatment is one of thecompositions set forth in Table 4, above.

In one embodiment, prior to nebulization of the aminoglycosidecomposition, about 70% to about 100% of the aminoglycoside present inthe composition is liposomal complexed. In a further embodiment, theaminoglycoside is an aminoglycoside. In even a further embodiment, theaminoglycoside is amikacin. In another embodiment, prior tonebulization, about 80% to about 99%, or about 85% to about 99%, orabout 90% to about 99% or about 95% to about 99% or about 96% to about99% of the aminoglycoside present in the composition is liposomalcomplexed. In a further embodiment, the aminoglycoside is amikacin ortobramycin. In even a further embodiment, the aminoglycoside isamikacin. In another embodiment, prior to nebulization, about 98% of theaminoglycoside present in the composition is liposomal complexed. In afurther embodiment, the aminoglycoside is amikacin or tobramycin. Ineven a further embodiment, the aminoglycoside is amikacin (e.g., asamikacin sulfate).

In one embodiment, upon nebulization, about 20% to about 50% of theliposomal complexed aminoglycoside agent is released, due to shearstress on the liposomes. In a further embodiment, the aminoglycosideagent is an amikacin. In another embodiment, upon nebulization, about25% to about 45%, or about 30% to about 40% of the liposomal complexedaminoglycoside agent is released from the liposomal complex, due toshear stress on the liposomes. In a further embodiment, theaminoglycoside agent is amikacin. In even a further embodiment, theamikacin is amikacin sulfate.

Upon nebulization of the composition described herein, i.e., foradministration to a patient in need of treatment of an NTM infection, anaerosolized composition is formed, and in one embodiment, the massmedian aerodynamic diameter (MMAD) of the aerosolized composition isabout 1.0 μm to about 4.2 μm as measured by the Anderson CascadeImpactor (ACI). In one embodiment, the MMAD of the aerosolizedcomposition is about 3.2 μm to about 4.2 μm as measured by the ACI. Inone embodiment, the MMAD of the aerosolized composition is about 1.0 μmto about 4.9 μm as measured by the Next Generation Impactor (NGI). In afurther embodiment, the MMAD of the aerosolized composition is about 4.4μm to about 4.9 μm as measured by the NGI.

The fine particle fraction (FPF) of the aerosolized composition, in oneembodiment, is greater than or equal to about 64%, as measured by theAnderson Cascade Impactor (ACI), or greater than or equal to about 51%,as measured by the Next Generation Impactor (NGI). In one embodiment,embodiment, the FPF of the aerosolized composition is greater than orequal to about 70%, as measured by the ACI, greater than or equal toabout 51%, as measured by the NGI, or greater than or equal to about60%, as measured by the NGI.

Upon nebulization, the liposomes in the pharmaceutical composition leakdrug. In one embodiment, the amount of liposomal complexedaminoglycoside post-nebulization is about 45% to about 85%, or about 50%to about 80% or about 51% to about 77%. These percentages are alsoreferred to herein as “percent associated aminoglycosidepost-nebulization.” As provided herein, in one embodiment, the liposomescomprise an aminoglycoside, e.g., amikacin. In one embodiment, thepercent associated aminoglycoside post-nebulization is from about 60% toabout 70%. In a further embodiment, the aminoglycoside is amikacin. Inanother embodiment, the percent associated aminoglycosidepost-nebulization is about 67%, or about 65% to about 70%. In a furtherembodiment, the aminoglycoside is amikacin. In even a furtherembodiment, the amikacin is amikacin sulfate.

In one embodiment, the percent associated aminoglycosidepost-nebulization is measured by reclaiming the aerosol from the air bycondensation in a cold-trap, and the liquid is subsequently assayed forfree and encapsulated aminoglycoside (associated aminoglycoside).

In another embodiment, the methods provided herein are implemented forthe treatment or prophylaxis of one or more NTM pulmonary infections ina cystic fibrosis patient. In a further embodiment, the compositionadministered to the patient in need of treatment is one of thecompositions set forth in Table 4, above.

In one embodiment, the patient in need of treatment of the NTM pulmonaryinfection is a bronchiectasis patient. In one embodiment, thebronchiectasis is non-Cystic Fibrosis (CF) bronchiectasis. In anotherembodiment, the bronchiectasis is associated with CF in a patient inneed of treatment.

In another embodiment, the patient in need of treatment of the NTMpulmonary infection is a COPD patient. In yet another embodiment, thepatient in need of treatment of the NTM pulmonary infection is an asthmapatient. In a further embodiment, the composition administered to thepatient in need of treatment is one of the compositions set forth inTable 4, above.

In one embodiment, a patient in need of treatment with one of themethods described herein is a Cystic Fibrosis patient, a bronchiectasispatient, a ciliary dyskinesia patient, a chronic smoker, a chronicobstructive pulmonary disorder (COPD) patient, or a patient who has beenpreviously non-responsive to treatment. In another embodiment, a cysticfibrosis patient is treated for an NTM pulmonary infection with one ofthe methods provided herein. In yet another embodiment, the patient is abronchiectasis patient, a COPD patient or an asthma patient. Thepulmonary NTM infection, in one embodiment, is MAC, M. kansasii, M.abscessus, or M. fortuitum. In a further embodiment, the pulmonary NTMinfection is a MAC infection.

A patient subjected to the methods described herein, in one embodiment,has a comorbid condition. For example, in one embodiment, the patient inneed of treatment with one of the methods described herein has diabetes,mitral valve disorder (e.g., mitral valve prolapse), acute bronchitis,pulmonary hypertension, pneumonia, asthma, trachea cancer, bronchuscancer, lung cancer, cystic fibrosis, pulmonary fibrosis, a larynxanomaly, a trachea anomaly, a bronchus anomaly, aspergillosis, HIV orbronchiectasis, in addition to the pulmonary NTM infection.

In one embodiment, a patient subjected to one of the NTM methodsdescribed herein exhibits an NTM culture conversion to negative duringthe administration period of the liposomal aminoglycoside composition,or after the administration period has concluded. The time toconversion, in one embodiment, is about 10 days, or about 20 days orabout 30 days or about 40 days, or about 50 days, or about 60 days, orabout 70 days, or about 80 days, or about 90 days, or about 100 days orabout 110 days. In another embodiment, the time to conversion is fromabout 20 days to about 200 days, from about 20 days to about 190 days,from about 20 days to about 180 days, from about 20 days to about 160days, from about 20 days to about 150 days, from about 20 days to about140 days, from about 20 days to about 130 days, from about 20 days toabout 120 days, from about 20 days to about 110 days, from about 30 daysto about 110 days, or from about 30 days to about 100 days.

In some embodiments, the patient experiences an improvement in lungfunction for at least 15 days after the administration period ends, ascompared to the FEV₁ of the patient prior to treatment. For example, thepatient may experience an increase in FEV₁, an increase in blood oxygensaturation, or both. In some embodiments, the patient has an FEV₁ (afterthe administration period or treatment cycle) that is increased by atleast 5% over the FEV₁ prior to the administration period. In otherembodiments, FEV₁ is increased by 5 to 50% over the FEV₁ prior to theadministration period. In other embodiments, FEV₁ is increased by 25 to500 mL over FEV₁ prior to the administration period. In someembodiments, blood oxygen saturation is increased by at least 1% overoxygen saturation prior to the administration period.

In one embodiment, the 6-minute walk test (6MWT) is used to assess theeffectiveness of the treatment methods provided herein. The 6MWT is usedfor the objective evaluation of functional exercise capacity and is apractical, simple test that measures the distance that a patient canwalk in a period of 6 minutes (see American Thoracic Society. (2002). AmJ Respir Crit Care Med. 166, pp. 111-117, incorporated by referenceherein in its entirety for all purposes).

In one embodiment, a patient subjected to one of the NTM methodsdescribed herein exhibits an increased number of meters walked in the6MWT, as compared to prior to undergoing the treatment method. Theincreased number of meters walked in the 6MWT, in one embodiment, isabout 5 meters, about 10 meters, about 15 meters, about 20 meters, about25 meters, about 30 meters, about 35 meters, about 40 meters, about 45meters, or about 50 meters. In another embodiment, the increased numberof meters walked in the 6MWT is at least about 5 meters, at least about10 meters, at least about 15 meters, at least about 20 meters, at leastabout 25 meters, at least about 30 meters, at least about 35 meters, atleast about 40 meters, at least about 45 meters, or at least about 50meters. In yet another embodiment, the increased number of meters walkedin the 6MWT is from about 5 meters to about 50 meters, or from about 5meters to about 40 meters, or from about 5 meters to about 30 meters orfrom about 5 meters to about 25 meters.

In another embodiment, a patient subjected to one of the NTM methodsdescribed herein exhibits a greater number of meters walked in the 6MWT,as compared to a patient undergoing a non-liposomal aminoglycosidetreatment. The greater number of meters walked in the 6MWT, as comparedto a patient undergoing a non-liposomal aminoglycoside treatment, in oneembodiment, is about 5 meters, about 10 meters, about 15 meters, about20 meters, about 25 meters, about 30 meters, about 35 meters, about 40meters, about 45 meters, about 50 meters, about 60 meters, about 70meters or about 80 meters. In another embodiment, the greater number ofmeters walked in the 6MWT is at least about 5 meters, at least about 10meters, at least about 15 meters, at least about 20 meters, at leastabout 25 meters, at least about 30 meters, at least about 35 meters, atleast about 40 meters, at least about 45 meters, or at least about 50meters. In yet another embodiment, the greater number of meters walkedin the 6MWT is from about 5 meters to about 80 meters, or from about 5meters to about 70 meters, or from about 5 meters to about 60 meters orfrom about 5 meters to about 50 meters.

In one embodiment, the liposomal aminoglycoside composition providedherein is administered to a patient in need of treatment of an NTM lungdisease with an additional therapy.

In one embodiment, the liposomal aminoglycoside composition providedherein is administered to a patient in need of treatment of an NTM lungdisease with one or more additional therapeutic agents. The one or moreadditional therapeutics agents in one embodiment, is administeredorally. In another embodiment, the one or more additional therapeuticsagents in one embodiment, is administered intravenously. In yet anotherembodiment, the one or more additional therapeutics agents in oneembodiment, is administered via inhalation.

The one or more additional therapeutic agents in one embodiment, is amacrolide antibiotic. In a further embodiment, the macrolide antibioticis azithromycin, clarithromycin, erythromycin, carbomycin A, josamycin,kitamycin, midecamycin, oleandomycin, solithromycin, spiramycin,troleandomycin, tylosin, roxithromycin, or a combination thereof. In afurther embodiment, the macrolide antibiotic is administered orally.

In one embodiment, the one or more additional therapeutic agents is themacrolide antibiotic azithromycin, clarithromycin, erythromycin, or acombination thereof. In a further embodiment, the macrolide antibioticis administered orally.

In another embodiment, the liposomal aminoglycoside composition providedherein is administered to a patient in need of treatment of an NTM lungdisease with one or more additional therapeutic agents, and the one ormore additional therapeutic agents is a rifamycin compound. In a furtherembodiment, the rifamycin is rifampin. In another embodiment, therifamycin is rifabutin, rifapentine, rifaximin, or a combinationthereof.

In yet embodiment, the one or more additional therapeutic agents is aquinolone. In a further embodiment, the quinolone is a fluoroquinolone.In another embodiment, the quinolone is ciprofloxacin, levofloxacin,gatifloxacin, enoxacin, levofloxacin, ofloxacin, moxifloxacin,trovafloxacin, or a combination thereof.

In one embodiment, a second therapeutic agent is administered to thepatient in need of NTM treatment, and the second therapeutic agent is asecond aminoglycoside. In a further embodiment, the secondaminoglycoside is amikacin, apramycin, arbekacin, astromicin,bekanamycin, boholmycin, brulamycin, capreomycin, dibekacin, dactimicin,etimicin, framycetin, gentamicin, H107, hygromycin, hygromycin B,inosamycin, K-4619, isepamicin, KA-5685, kanamycin, neomycin,netilmicin, paromomycin, plazomicin, ribostamycin, sisomicin,rhodestreptomycin, sorbistin, spectinomycin, sporaricin, streptomycin,tobramycin, verdamicin, vertilmicin, a pharmaceutically acceptable saltthereof, or a combination thereof. In a further embodiment, the secondaminoglycoside is administered intravenously or via inhalation. In oneembodiment the second aminoglycoside is streptomycin.

In another embodiment, the liposomal aminoglycoside composition providedherein is administered to a patient in need of treatment of an NTM lungdisease with one or more additional therapeutic agents, and the one ormore additional therapeutic agents is ethambutol, isoniazid, cefoxitinor imipenem.

EXAMPLES

The present invention is further illustrated by reference to thefollowing Examples. However, it should be noted that these Examples,like the embodiments described above, are illustrative and are not to beconstrued as restricting the scope of the invention in any way.

Example 1: Randomized-Double Blind Study of Liposomal Amikacin forInhalation (LAI) in Patients with Non-Tuberculous Mycobacterium (NTM)Lung Disease (LD)

The increasing prevalence of NTM-LD is a public health concern and itsmanagement, particularly in cystic fibrosis patients, is complicated byprolonged use of multidrug regimens, drug toxicity, and poor responserates. LAI (also referred to herein as “Arikayce™” or “ARIKAYCE™”) is asustained-release lipid composition of amikacin in development fortreatment of patients with recalcitrant NTM lung disease. This studyevaluated the efficacy, safety, and tolerability of LAI in thesepatients in a randomized, double-blind (DB) study, conducted at 19centers in North America. FIG. 1 is a flow chart showing the studydesign and FIG. 2 shows the patient distribution for the study.

The LAI composition had the following components:

LAI composition Amikacin Sulfate ~70 mg/mL DPPC ~30-35 mg/mL Cholesterol~15-17 mg/mL NaCl ~1.5%

Eligible NTM patients on a stable drug regimen were stratified based onpresence or absence of cystic fibrosis (CF), and Mycobacterium aviumcomplex (MAC) versus Mycobacterium abscessus (M. abscessus) lungdisease, and randomized 1:1 to receive either once daily 590 mg LAI orplacebo via eFlow® nebulizer system (PART Pharma GmbH) for 84 days addedto their ongoing stable drug regimen. FIG. 3 shows the number ofpatients in each group (randomized per strata). Patients were eligiblefor enrollment if they had pulmonary NTM infection refractory toAmerican Thoracic Society/Infectious Disease Society of America(ATS/IDSA) guideline-based therapy for months prior to screening.

After completing the double blind (DB) phase, patients who consented tothe open-label (OL) phase received LAI 590 mg once daily, for 84 moredays (FIGS. 1 and 2).

Of 136 screened patients, 90 were randomized (19% CF; 81% non-CF; 64%with MAC and 36% with M. abscessus). 54% of patients were >60 years ofage; 31% were >40-60 years, and 14% were 18-40 years. The baseline meanage was 58.5 years (standard deviation, 15.83 years).

The study is complete, with 80 and 59 patients having completed the DBand OL phases, respectively. Demographics and baseline characteristicsof the mITT population are provided below in Table 5.

TABLE 5 Demographics and Baseline Characteristics of mITT Population LAIPlacebo Overall (n = 44) (n = 45) (n - 89) Gender, n (%) Male  6 (13.6) 5 (11.1) 11 (12.4) Female 38 (86.4) 40 (88.9) 78 (87.6) Race/Ethnicity,n (%) Caucasian (not of 42 (95.5) 40 (88.9) 82 (92.1) Hispanic Origin)Hispanic 0 2 (4.4) 2 (2.2) African 0 1 (2.2) 1 (1.1) Asian 2 (4.5) 2(4.4) 4 (4.5) Other 0  0  0 Baseline Age, years n 44  45 89 Mean (SD)58.0 (16.61)  59.1 (15.20)  58.5 (15.83)  Median  61.5   63.0   63.0Min, Max 18, 85  19, 80  18, 85  Baseline FEV₁ Percent Predicted n 44 45 89 Mean (SD) 65.56 (21.339)  62.56 (17.168)  63.06 (19.239)  Median  61.25   61.00   61.00 Min, Max 30.2, 114.9 34.4, 101.6 30.2, 114.9

The sample population enrolled in the mITT study exhibited thefollowing: (1) comorbid lung disease, with 17 of the patients havingcystic fibrosis; (2) a mean age of 59 years, including the youngercystic fibrosis patients; (3) lung abnormalities including 68 patientswith cavitary lesions, and 21 patients with nodular disease whichfurther includes minimal cavitary disease; (4) a mean body mass index(BMI) of 21.98, whereas comparable CDC data collected from between 2007and 2010 reveals U.S average BMI of adult males to be 28.6 and adultfemales to be 28.7; and (5) an average baseline of ˜441 m for allpatients, with both arms having approximately the same mean baselinesix-minute walk distance.

Sputum for semi-quantitative mycobacterial culture, smear status,signs/symptoms, pulmonary exacerbation occurrence, antimycobacterialdrug rescue, six-minute walk distance (6MWD), computed tomography of thechest, spirometry, clinical/laboratory safety parameters, and quality oflife measures were evaluated every 28 days. The primary endpoint waschange from baseline on the semi-quantitative scale for mycobacterialculture; a secondary endpoint was the proportion of patients with NTMculture conversion to negative for LAI vs placebo at Day 84. Allpatients had a safety follow-up visit 28 days after the last dose ofstudy drug, up to Day 196 for those in the OL phase.

FIG. 4 is a graph showing the mean change from baseline on the full semiquantitative scale for mycobacterial culture (mITT population) as afunction of study day in both the double-blind phase and the open-labelphase of the study. As shown in the figure, patients treated with LAIshowed at least a one-step reduction in the treatment arm versus theplacebo arm in the double-blind phase.

The proportion of patients with negative sputum cultures for NTM in eachsubgroup by treatment arm at Day 84 and Day 168 (mITT population) aresummarized in Tables 6-8. At Day 84, statistically significantbetween-group differences in patients achieving negative sputum culturesfor NTM, in favor of LAI vs. placebo, were seen in patients with non-CFinfection (P=0.01), MAC infection (P=0.017), females (P=0.004),Caucasians (P=0.031), and patients aged <63 years (P=0.041) (Table 6).

At Day 168, statistically significantly more patients with MAC infectionin the prior LAI arm vs. prior placebo arm had negative sputum culturesfor NTM (P=0.026) (Table 6). In subgroup analyses (Table 7 and Table 8)of patients with NTM lung infection refractory to guideline-basedtherapy, LAI appeared superior to placebo with regard to negative sputumcultures for NTM in patients with non-CF underlying lung disease and MACinfection. The subgroup of patients with non-CF MAC infectiondemonstrated a positive efficacy result within the timeframe of thestudy (i.e., 12-week double-blind phase and 12-week open-label phase)

Time to culture conversion showed statistically significantly greaterproportion of patients in the LAI arm becoming culture negative at allvisits in the double blind phase (Days 28, 56, and 84) (FIG. 5 top).Specifically, LAI achieved statistical significance in achieving anegative culture at Day 84, with 11 of 44 patients on LAI versus 3 of 45patients on placebo (P=0.01) (FIG. 5 top). Compared with placebo, LAIdemonstrated statistical significance with regard to the proportion ofpatients with MAC infections who achieved culture negativity at Day 56(LAI, 10/29 patients vs. placebo, 2/28 patients; P=0.0144) and at Day 84(LAI, 10/29 patients vs, placebo, 3/28 patients; P=0.0273) (FIG. 5bottom).

In patients refractory to NTM-regimens for at least 6 months, LAI, aninhaled amikacin composition, lead to significantly greater cultureconversion compared to placebo within 84 days. Patients with at leastone NTM culture negative result are provided in FIG. 6.

TABLE 6 Proportion of Patients with negative sputum cultures for NTM ineach subgroup by treatment arm at days 84 and 168 (mITT population)^(a)Day 84 Day 168 (double-blind phase) (open-label phase) Subgroups, LAIPlacebo Prior LAI^(c) Prior placebo^(c) n/n (%) (n = 44) (n = 45) Pvalue^(b) (n = 35) (n = 43) P value^(b) Infection type MAC 10/27 (37.0)3/28 (10.7) .017 12/24 (50.0) 6/27 (22.2) .026 MAB 1/14 (7.1) 0/17 .3171/11 (9.1) 2/14 (14.3) .691 CF 0/7 0/9  NA   1/6 (16.7) 0/7 .221 Non-CF11/34 (32.4) 3/36 (8.3)  .01 12/29 (41.4) 8/34 (23.5) .122 Gender Female 11/36 (30.60) 2/40 (5.0)  .004 12/31 (38.7) 8/36 (22.2) .137 Male 0/5 1/5 (20.0) .414   1/4 (25.0) 0/5 .480 Ethnicity Caucasian 10/39 (25.6)3/40 (7.5)  .031 13/33 (39.4) 8/37 (21.6) .107 Non-Caucasian  1/2 (50.0)0/5  NA 0/2 0/4 N/A Age <63 years  7/21 (33.3) 2/22 (9.1)  .041  7/19(36.8) 3/20 (15.0) .098 >63 years  4/20 (20.0) 1/23 (4.3)  .108  6/16(37.5) 5/21 (23.8) .367 CF, cystic fibrosis; LAI, liposomal amikacin forinhalation; MAB, Mycobacterium avium complex; mITT, modifiedintent-to-treat; NTM, nontuberculous mycobacteria; NA, not available.^(a)Missing values are excluded under the assumption of missing atrandom, for which missing baseline or post-baseline values are excludedbut all non-missing data are included (ie, exclusion is not atsubject-level but, rather, at time point-level). ^(b)For pairwisecomparisons of the LAI arm with the placebo arm, a stratifiedCochran-Mantel-Haenszel test of treatment arm adjusting for therandomization strata was used. ^(c)All patients received LAI in theopen-label phase.

TABLE 7 Subgroup analysis of patients with MAC infection who achievednegative sputum cultures for NTM by treatment arm at days 84 and 168 168(mITT population)^(a) Day 84 Day 168 (double-blind phase) (open-labelphase) Subgroups, n/n LAI Placebo Prior LAI^(c) Prior placebo^(c) (%) (n= 29) (n = 28) P value^(b) (n = 24) (n = 28) P value^(b) Infection typeCF 0/2 0/1 NA 0/2 0/1 N/A Non-CF 10/25 (40.0) 3/27 (11.1) .025 12/22(54.6) 6/26 (23.1) .037 Cavitary disease  5/17 (29.4) 2/20 (10.0) .212 5/14 (35.7) 2/19 (10.5) .106 Non-cavitary  5/10 (50.0)  1/8 (12.5) .152 7/10 (70.0)  4.8 (50.0) .631 disease Gender Female 10/25 (40.0) 2/25(8.0)  .018 12/22 (54.6) 6/24 (25.0) .069 Male 0/2  1/3 (33.3) 1.000 0/20/3 N/A Ethnicity Caucasian 10/27 (37.0) 3/25 (12.0) .055 12/24 (50.0)6/24 (25.0) .135 Non-Caucasian 0/0 0/3 NA 0/0 0/3 NA Age <63 years  6/13(46.2) 2/11 (18.2) .211  6/13 (46.2) 2/11 (18.2) .211 >63 years  4/14(28.6) 1/17 (5.9)  .148  6/11 (54.6) 4/16 (25.0) .224 CF, cysticfibrosis; LAI, liposomal amikacin for inhalation; MAC, Mycobacteriumavium complex; mITT, modified intent-to-treat; NA, not available.^(a)Missing values are excluded under the assumption of missing atrandom, for which missing baseline or post-baseline values are excludedbut all non-missing data are included (ie, exclusion is not atsubject-level but, rather, at time point-level). ^(b)Pairwisecomparisons of the LAI arm with the placebo arm were based on Fisher'sExact Test. ^(c)All patients received LAI in the open-label phase.

TABLE 8 Subgroup analysis of patients with M. abscessus (MAB) infectionwho achieved negative sputum cultures for NTM by treatment arm at days84 and 168 168 (mITT population)^(a) Day 84 Day 168 (double-blind phase)(open-label phase) Subgroups, n/n LAI Placebo Prior LAI^(c) Priorplacebo^(c) (%) (n = 15) (n = 17) P value^(b) (n = 11) (n = 15) Pvalue^(b) Infection type CF 0/5 0/8 NA 1/4 (25.0) 0/6 400 Non-CF  1/9(11.1) 0/9 1.000 0/7  2/8 (25.0) .467 Cavitary disease 1/13 (7.7)  0/15.464 1/10 (10.0)  2/12 (16.7) 1.000 Non-cavitary 0/1 0/2 NA 0/1 0/2 N/Adisease Gender Female 1/11 (9.1)  0/15 .423 0/9 212 (16.7) .486 Male 0/30/2 NA 1/2 (50.0) 0/2 1.000 Ethnicity Caucasian  0/12  0/15 NA 1/9(11.1) 2/13 (15.4) 1.000 Non-Caucasian  1/2 (50.0) 0/2 1.000 0/2 0/1 NAAge <63 years  1/8 (12.5)  0/11 .421 1/6 (16.7)  1/9 (11.1) 1.000 >63years 0.6 0/6 NA 0/5  1/5 (20.0) 1.000 CF, cystic fibrosis; LAI,liposomal amikacin for inhalation; MAB, Mycobacterium abscessus; MITT,modified intent-to-treat; NA, not available. ^(a)Missing values areexcluded under the assumption of missing at random, for which missingbaseline or post-baseline values are excluded but all non-missing dataare included (ie, exclusion is not at subject-level but, rather, at timepoint-level). ^(b)Pairwise comparisons of the LAI arm with the placeboarm were based on Fisher's Exact Test. ^(c)All patients received LAI inthe open-label phase.

The six-minute walk test (6MWT) assessed the impact of LAI on overallphysical function or capacity. Results for the 6MWT endpoint (changefrom baseline from Day 1 to Day 84 at end of double blind study) areprovided in FIG. 7 and FIG. 8. LAI demonstrated statistical significancein the 6MWT in the double-blind phase (LAI vs placebo: 23.895 vs −25.032meters, P=0.009). The mean change from baseline to Day 84 in distancewalked (meters) in the 6MWT was significantly higher for patientsreceiving LAI vs. placebo (20.64 m vs. −25.03 m) (FIG. 7 bottom). In theopen-label phase, patients in the LAI arm continued to improve on the6MWT and patients in the placebo group who started LAI showed a dramaticdecline in the rate of deterioration (FIGS. 7 and 8). Further, asignificant difference was seen in the mean change from baseline to Day168 in the 6MWT score for patients with sustained culture-negativestatus to the end of the open-label phase vs. those without sustainedculture-negative status (55.75 m vs. −13.42 m) (FIG. 8 bottom).

Patients with NTM lung infections refractory to treatment showedimprovement in distance walked in the 6MWT when LAI was added to theirbackground of guideline-based therapy. Patients with sustainedculture-negative status during the study achieved better physicalfunctional capacity as assessed by the 6MWT.

The sample population enrolled in the mITT study exhibited thefollowing, prior to day 168, with regard to culture conversion, measuredas three consecutive negative sputum cultures: (1) a total of 16patients demonstrated culture conversion, all of which were non-cysticfibrosis; (2) 15 patients had MAC and 1 had M. abscessus; (3) 8 patientsexhibited no treatment success despite greater than 24 months of non-LAItreatment methods, 4 patients exhibited no treatment success despite 12to 24 months of non-LAI treatment methods, and 4 patients exhibited notreatment success despite 6 to 12 months of non-LAI treatment methods;(4) 7 patients exhibited nodular disease, 2 patients exhibited nodulardisease and minimal cavitary lesions, and 7 patients exhibited cavitarylesions; (5) 11 patients started to convert at or prior to day 56 afterbeginning LAI treatment methods, 2 patients converted at day 84 afterbeginning LAI treatment methods, and 3 patients converted at day 112after beginning LAI treatment methods; and (6) 6MWT for converters(n=16) vs. nonconverters (n=43) at day 168 was 89.34 meters (converters)vs. 3.85 meters (nonconverters), with a p-value of 0.0034.

No difference between arms in patients with hemoptysis, tinnitus, andhearing loss was found.

Moreover, it was found that patients entering the open label phase fromLAI in the double blind phase (see FIG. 1 for study design) continued toimprove. Additionally, patients entering open label phase from placebodemonstrate a dramatic decrease in their rate of decline. Most treatmentemergent adverse events (TEAEs) were mild or moderate in severity, andthe majority of TEAEs were respiratory in nature (Table 9). Local eventsand infective exacerbation of the underlying lung disease were the mostcommon TEAEs. Few patients discontinued the study drug due to theseevents.

TABLE 9 Overview of Adverse Events Through End of Open-label Phase(Safety Population) Double Blind Phase^(a) Open-Label Phase^(b) LAIPlacebo LAI^(c) Placebo^(c) (n = 44) (n = 45) (n = 35) (n = 43) Subjectswith treatment-emergent 41 (93.2) 40 (88.9) 31 (88.6) 42 (97.7) adverseevents (TEAEs), n(%) TEAEs, n 240 140  107 160  Subjects with TEAEs bymaximum severity, n (%) Grade 1: Mild 12 (27.3) 25 (55.6) 16 (45.7) 10(23.3) Grade 2: Moderate 24 (54.5) 10 (22.2) 10 (28.6) 24 (55.8) Grade3: Severe 4 (9.1)  5 (11.1)  4 (11.4)  8 (18.6) Grade 4:Life-threatening or disabling  0 0  0 0 Grade 5: Death^(d) 1 (2.3) 0 1(2.9) 0 Subjects with TEAEs by seriousness, n (%) Serious  8 (18.2) 4(8.9)  5 (14.3)  5 (11.6) Not serious 33 (75.0) 36 (80.0) 26 (74.3) 37(86.0) Treatment-emergent serious adverse events, n  12 5  10 5 Subjectswith TEAEs by relationship to study drug, n (%) Related 3 (6.8) 0 17(48.6) 26 (60.5) Not related  5 (11.4) 4 (8.9) 14 (40.0) 16 (37.2)Subjects with treatment-emergent  5 (11.4)  5 (11.1) 2 (5.7) 2 (4.7)audiovestibular adverse events, n (%) Subjects with treatment-emergentrenal adverse  1(2.3) 0 1 (2.9) 0 events, n(%) Subjects with adverseevents leading to study  8 (18.2) 0  6 (17.1) 12 (27.9) drugdiscontinuation, n (%)

Example 2: Study of Liposomal Amikacin for Inhalation (LAI) in Patientswith Non-CF M. avium Complex (MAC) Lung Infection

LAI (also referred to herein as “Arikayce™” or “ARIKAYCE™”) is asustained-release lipid composition of amikacin in development fortreatment of patients with recalcitrant NTM lung disease. In this study,the efficacy, safety, and tolerability of LAI is assessed in non-CysticFibrosis patients having M. avium complex (MAC) lung infection. FIG. 9is a flow chart showing the study design.

The LAI composition has the following components:

LAI composition Amikacin Sulfate ~70 mg/mL DPPC ~30-35 mg/mL Cholesterol~15-17 mg/mL NaCl ~1.5%

Table 10 provides the inclusion criteria for the study.

TABLE 10 Inclusion Criteria for Study Age ≥ 18 years ≤ 85 yearsDiagnosis of pulmonary NTM MAC lung disease Failed prior treatmentMulti-drug regimen for at least 6 months; last dose within the prior 12months

Patients are randomized 2:1 into two groups: (i) 590 mg LAI+backgroundtherapy and (ii) background therapy only). Each patient group issubjected to daily dosing for 8 months. Primary culture conversion isassessed at 6 months. 6MWT is also carried out for each patient at 6months.

Culture converters continue treatment for 12 months post conversion.

All, documents, patents, patent applications, publications, productdescriptions, and protocols which are cited throughout this applicationare incorporated herein by reference in their entireties for allpurposes.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Modifications and variationof the above-described embodiments of the invention are possible withoutdeparting from the invention, as appreciated by those skilled in the artin light of the above teachings. It is therefore understood that, withinthe scope of the claims and their equivalents, the invention may bepracticed otherwise than as specifically described. Accordingly, theforegoing descriptions and drawings are by way of example only and thedisclosure is described in detail by the claims that follow.

The invention claimed is:
 1. A method for treating a Mycobacterium aviumcomplex (MAC) lung infection, comprising: administering to the patient amacrolide antibiotic; and administering to the lungs of the patient foran administration period of at least six months, a pharmaceuticalcomposition comprising amikacin, or a pharmaceutically acceptable saltthereof, encapsulated in a plurality of liposomes, wherein the lipidcomponent of the plurality of liposomes consists of an electricallyneutral phospholipid and cholesterol, wherein administering to the lungsof the patient comprises aerosolizing the pharmaceutical composition viaa nebulizer to provide an aerosolized pharmaceutical compositioncomprising a mixture of free amikacin, or a pharmaceutically acceptablesalt thereof, and liposomal complexed amikacin, or a pharmaceuticallyacceptable salt thereof, and administering the aerosolizedpharmaceutical composition via the nebulizer to the lungs of the patientonce daily in a single dosing session during the administration period;wherein during the administration period or subsequent to theadministration period, the patient exhibits an NTM culture conversion tonegative and walks an increased number of meters in the 6 minute walktest (6MWT), as compared to the number of meters walked by the patientprior to undergoing the treatment method.
 2. The method of claim 1,wherein prior to the treating, the patient was unresponsive to adifferent MAC therapy.
 3. The method of claim 1, prior to the treating,the patient was unresponsive to an American Thoracic Society/InfectiousDisease Society of America (ATS/IDSA) MAC guideline-based therapy (GBT).4. The method of claim 3, wherein prior to the treating, the patient wasunresponsive to the ATS/IDSA MAC GBT for at least 6 months.
 5. Themethod of claim 1, wherein the macrolide antibiotic is azithromycin,clarithromycin, erythromycin, carbomycin A, josamycin, kitamycin,midecamycin, oleandomycin, solithromycin, spiramycin, troleandomycin,tylosin, roxithromycin, or a combination thereof.
 6. The method of claim5, wherein the macrolide antibiotic is clarithromycin.
 7. The method ofclaim 5, wherein the macrolide antibiotic is azithromycin.
 8. The methodof claim 1, further comprising administering to the patient a rifamycincompound.
 9. The method of claim 8, wherein the rifamycin compound isrifampin or rifabutin.
 10. The method of claim 3, further comprisingadministering to the patient a rifamycin compound.
 11. The method ofclaim 4, further comprising administering to the patient a rifamycincompound.
 12. The method of claim 11, wherein the rifamycin compound isrifampin or rifabutin.
 13. The method of claim 1, further comprisingadministering to the patient ethambutol.
 14. The method of claim 3,further comprising administering to the patient ethambutol.
 15. Themethod of claim 4, further comprising administering to the patientethambutol.
 16. The method of claim 8, further comprising administeringto the patient ethambutol.
 17. The method of claim 10, furthercomprising administering to the patient ethambutol.
 18. The method ofclaim 11, further comprising administering to the patient ethambutol.19. The method of claim 12, further comprising administering to thepatient ethambutol.
 20. The method of claim 1, wherein the amikacin orpharmaceutically acceptable salt thereof is amikacin sulfate.
 21. Themethod of claim 3, wherein the amikacin or pharmaceutically acceptablesalt thereof is amikacin sulfate.
 22. The method of claim 8, wherein theamikacin or pharmaceutically acceptable salt thereof is amikacinsulfate.
 23. The method of claim 18, wherein the amikacin orpharmaceutically acceptable salt thereof is amikacin sulfate.
 24. Themethod of claim 1, wherein the electrically neutral phospholipid is anelectrically neutral phosphatidylcholine.
 25. The method of claim 20,wherein the electrically neutral phosphatidylcholine isdipalmitoylphosphatidylcholine (DPPC).
 26. The method of claim 23,wherein the electrically neutral phospholipid isdipalmitoylphosphatidylcholine (DPPC).
 27. The method of claim 1,wherein during the single dosing session, the aerosolized pharmaceuticalcomposition is administered in less than about 15 minutes.
 28. Themethod of claim 1, wherein during the single dosing session, theaerosolized pharmaceutical composition is administered in about 10minutes to about 14 minutes.
 29. The method of claim 1, wherein theincreased number of meters walked by the patient is at least about 5meters.
 30. The method of claim 1, wherein the increased number ofmeters walked by the patient is at least about 20 meters.
 31. The methodof claim 1, wherein the increased number of meters walked by the patientis at least about 25 meters.
 32. The method of claim 18, wherein theincreased number of meters walked by the patient is at least about 20meters.
 33. The method of claim 19, wherein the increased number ofmeters walked by the patient is at least about 25 meters.
 34. The methodof claim 1, wherein the increased number of meters walked by the patientis from about 5 meters to about 50 meters.
 35. The method of claim 3,wherein the increased number of meters walked by the patient is fromabout 5 meters to about 50 meters.
 36. The method of claim 4, whereinthe increased number of meters walked by the patient is from about 5meters to about 50 meters.
 37. The method of claim 8, wherein theincreased number of meters walked by the patient is from about 5 metersto about 50 meters.
 38. The method of claim 11, wherein the increasednumber of meters walked by the patient is from about 5 meters to about50 meters.
 39. The method of claim 14, wherein the increased number ofmeters walked by the patient is from about 5 meters to about 50 meters.40. The method of claim 15, wherein the increased number of meterswalked by the patient is from about 5 meters to about 50 meters.
 41. Themethod of claim 16, wherein the increased number of meters walked by thepatient is from about 5 meters to about 50 meters.
 42. The method ofclaim 17, wherein the increased number of meters walked by the patientis from about 5 meters to about 50 meters.
 43. The method of claim 18,wherein the increased number of meters walked by the patient is fromabout 5 meters to about 50 meters.
 44. The method of claim 19, whereinthe increased number of meters walked by the patient is from about 5meters to about 50 meters.
 45. The method of claim 23, wherein theincreased number of meters walked by the patient is from about 5 metersto about 50 meters.
 46. The method of claim 26, wherein the increasednumber of meters walked by the patient is from about 5 meters to about50 meters.